Casein kinase I (CKI) is a member of a family of ser/thr protein kinases which are ubiquitous to all eukaryotic cells. We have determined that 34 kDa CKI is conserved in sequence and size from human to yeast. In yeast, protein kinases homologous to the 34 kDa CKI are involved in growth regulation. The role that the 34 kDa CKIs play within higher eukaryotes is not known, but phosphatidylinositol-4,5-bisphosphate (PIP2) has been shown to regulate assembly and activity of erythroid 34 kDa CKI on plasma membranes and a yeast 34 kDa CKI is also inhibited by PIP2, suggesting that a 34 kDa CKI is structurally and functionally conserved throughout eukaryote evolution. Recently, a protein component has been partially purified which binds the 34 kDa CKI (CKI regulatory component "CRC") and antagonizes PIP2 inhibition in solution and on membranes. This component may be crucial for regulation of CKI membrane assembly, intracellular location, and kinase activity. Objectives: 1) Characterize PIP2 regulation of the 34 kDa CKI in vitro and in vivo. Using erythrocyte membranes as a model we will complete studies on PIP2 regulation of CKI membrane assembly. Using agonists which induce PIP2 turnover we will determine if CKI is activated by assaying immunoprecipitates of cell lysates, or reorganized within the cells using immunofluorescence. 2) A major objective is to isolate CRC and study CRC regulation of the 34 kDa CKI. Using purified CRC and CKI the interaction of CRC with CKI and with membranes will be studied in vitro. Using antibodies to CRC, the intracellular location will be studied upon agonist stimulation to see if this corresponds with the 34 kDa CKI location. 3) As CKI is a phosphoprotein, it will be determined (by 2-D phosphopeptide mapping) if phosphorylation of CKI in vivo is stimulated by growth factors or agonists such as bombesin. Does phosphorylation modulate activity, PIP2 regulation or interactions with CRC? If CRC is a phosphoprotein, it will be determined if phosphorylation of CRC occurs in response to agonists and the effect of phosphorylation on CKI-CRC interaction. Phosphorylation studies will be done in vivo in response to agonists and in vitro with purified protein kinases. 4) The cDNA for the erythroid 34 kDa CKI and CRC will be cloned and sequenced. In the long term this will be important for studying CRC interactions with other isoforms of CKI. Studies focused on the regulation of the CKIs will lead to a better understanding of metabolic regulation and cell growth; CKI may be another class of protein kinases which regulate certain aspects of cell proliferation or differentiation, and thus is likely to be important in the etiology of many disease states.